It is now well established that an elevated cholesterol level in blood is an indicator of a higher risk of coronary heart disease (CHD). Therefore, there is an increased demand for measuring blood cholesterol levels for screening general populations and for monitoring patients who are on cholesterol reducing diets or drugs. Since the recent data from the College of American Pathologists (CAP) survey of 5,000 participating laboratories revealed that a great deal of inaccuracy exists in reporting of cholesterol test results, it has become evident that there is an urgent need for an improved cholesterol test which will yield accurate and reliable results (Naito, H.K. and Hartmann, A.E., "The ABC's of Cholesterol Standardization", by the College of American Pathologists, 1987).
Conventionally, cholesterol is measured by direct or indirect chemical methods (U.S. Pat. Nos. 3,001,950; 3,479,154; 3,558,516) which involve the handling of highly corrosive material. Therefore, direct enzymatic methods today have virtually replaced the chemical methods in clinical laboratories. These enzymatic methods involve a) hydrolysis of cholesterol esters by an esterase or a combination of lipase and esterase (U.S. Pat. Nos. 3,776,816; 3,884,764; 3,925,164), b) subsequent oxidation of cholesterol by cholesterol oxidase (U.S. Pat. No. 3,907,645) which uses oxygen and produces cholestenone and hydrogen peroxide and c) quantitative measuring of hydrogen peroxide thus produced by means of peroxidase and reduced chromogen as shown below: ##STR1##
Alternatively, instead of measuring the hydrogen peroxide produced, the cholestenone produced or the oxygen consumed can be measured in this reaction to determine the level of cholesterol present in the sample. This enzymatic approach has admittedly brought considerable advancement in the determination of cholesterol due to its specificity, sensitivity and convenience when compared with previously used chemical methods. The process, however, has not been suitable for a rapid, kinetic (rate) method.
Cholesterol oxidases catalyze the most specific part of the reaction sequence in the determination of cholesterol. These cholesterol oxidases are obtained from organisms such as Nocardia, Brevibacterium or Streptomyces (U.S. Pat. Nos. 3,907,642; 3,983,005; 4,212,938; 4,226,713; 4,229,527; 4,350,762) and have a low Km (Michaelis and Menton) value of 10.sup.-4 to 10.sup.-5 M for cholesterol. The low Km values of these cholesterol oxidases do not allow the reaction to proceed as first order or pseudo-first order at a concentration range of up to 13 mmoles/L cholesterol, which is the range of interest for serum concentration. Therefore, the enzymatic methods developed with these cholesterol oxidases are "end-point" methods where all of the cholesterol is titrated by an excess of cholesterol oxidase. The "end-point" methods generally take 6 to 10 minutes to perform, require sample blanks and are subject to interferences by turbidity, bilirubin, hemolysis, ascorbate, and so forth. Some of these problems can be avoided and the cholesterol measurement further improved by developing "kinetic" or "rate" methods as an alternative to "end-point" methods. However, to develop a rapid and convenient kinetic" method for the cholesterol determination, a first order or pseudo-first order reaction is required. This can only be achieved if one has available a cholesterol oxidase with a Km of 10.sup.-2 to 10.sup.-3 M for cholesterol, i.e., a Km of about 20-100 fold larger than the substrate concentration of interest, thus making the reaction rate limiting. In this case the reaction will follow pseudo-first order kinetics.
The kinetic method thus developed can offer many advantages over end point methods, such as, 1) reduction in analysis time; 1-2 minutes, 2) less interference by turbidity, bilirubin, hemolysis and ascorbate, 3) obviate the need for a sample blank, and 4) offers improved accuracy.
Such a kinetic method for cholesterol determination was successfully developed by Deeg, et al. (U.S. Pat. No. 4,503,144) where the inventors were able to artificially increase the Km of cholesterol oxidase from genus Streptomyces which has a Km of 10.sup.-4 to 10.sup.-5 moles/L by adding a competitive inhibitor, such as, 3,4-dichlorophenol. By manipulating the concentration of 3,4-dichlorophenol and cholesterol oxidase, it was possible to increase the Km of the enzyme allowing the cholesterol oxidase reaction to proceed as pseudo-first order.
In contrast to Deeg et al., the present invention utilizes a new and unique cholesterol oxidase (GDS-41) isolated from a mutant of genus Nocardia which was found surprisingly to have an inherent Km value of 10.sup.-2 to 10.sup.-3 M for cholesterol. Therefore, it is the object of the present invention to provide a kinetic method and a test composition for the determination of cholesterol using this cholesterol oxidase. This invention provides a significant advantage over the previously described Deeg, et al. method as it does not require artificially increasing the Km value by using a competitive inhibitor and therefore, requires only about one tenth to one hundredth the amount of cholesterol oxidase normally used in a prior art test composition. As cholesterol oxidase is usually the most expensive ingredient in the test composition, this method will allow the development of a more cost effective cholesterol test. In addition, the method allows measuring the reaction rate within 0.5-1.5 minutes, thus permitting higher throughput using automatic clinical analyzers or alternatively, providing immediate results for use in "on-site" or physicians office testing.